The present invention relates to methods and compositions for performing biological assays. Specifically, the invention provides compositions, referred to herein as microstructures, which may be used in micro-fabricated devices for a variety of biological applications, including nucleic acid sequence analysis and polynucleotide amplification, as well as other biological assays and reactions.
Many techniques and assays have been developed for the analysis of biological samples. Practical applications of these techniques include the diagnosis of genetic diseases, the diagnosis of infectious diseases, forensic techniques, paternity determination, and genome mapping.
For example, in the field of nucleotide sequence analysis, many techniques have been developed to analyze nucleic acid sequences and detect the presence or absence of various genetic elements such as genetic mutations, and polymorphisms such as single nucleotide polymorphisms(hereinafter xe2x80x9cSNPsxe2x80x9d), base deletions, base insertions, and heterozygous as well as homozygous polymorphisms.
Currently, the most definitive method for analyzing nucleic acid sequences is to determine the complete nucleotide sequence of each nucleic acid segment of interest. Examples of how sequencing has been used to study mutations in human genes are included in the publications of Engelke et al. (1998, Proc. Natl. Acad. Sci. U.S.A. 85:544-548) and Wong et al. (1997, Nature 300:384-386). The most commonly used methods of nucleic acid sequencing include the dideoxy-mediated chain termination method, also known as the xe2x80x9cSanger Methodxe2x80x9d (Sanger, F. et al., 1975, J. Molec. Biol. 94:441; Porbe, J. et al., 1997, Science 238:336-340) and the chemical degradation or xe2x80x9cMaxam-Gilbertxe2x80x9d method (Maxam, A. M. et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:560).
Restriction fragment length polymorphism (hereinafter xe2x80x9cRFLPxe2x80x9d) mapping is another commonly used screen for DNA polymorphisms arising from DNA sequence variation. RFLP consists of digesting DNA with restriction endonucleases and analyzing the resulting fragments, as described by Botstein et al. (1980, Am. J. Hum. Genet. 32:314-331) and by White et al. (1998, Sci. Am. 258:40-48). Mutations that affect the sequence recognition of the endonuclease will alter enzymatic cleavage at that site, thereby altering the cleavage pattern of the DNA. DNA sequences are compared by looking for differences in restriction fragment lengths.
However, sequencing techniques such as the Sanger and Maxam-Gilbert methods involve series of nested reactions which are then analyzed on electrophoretic gels. RFLP analysis also requires analysis of reaction products on Southern Blots. These techniques can therefore be cumbersome to perform and analyze.
Alternative, simpler, and less cumbersome methods to analyze and/or sequence nucleic acid molecules have also been proposed. For example, there is considerable interest in developing methods of de novo sequencing using solid phase arrays (see, e.g., Chetverin, A. B. et al., 1994, Biotech. 12:1093-1099; Macevicz, U.S. Pat. No. 5,002,867; Beattie, W. G. et al., 1995, Molec. Biotech. 4:213-225; Drmanac, R. T., EP 797683; Church et al., U.S. Pat. No. 5,149,625; Gruber, L. S., EP 787183; each of which is incorporated herein by reference in its entirety) including universal sequencing arrays such as those described, e.g., by Head, S. et al. (U.S. patent application Ser. No. 08/976,427, filed Nov. 21, 1997, which is incorporated herein by reference in its entirety) and by Boyce-Jacino, M. et al. (U.S. patent application Ser. No. 09/097,791, filed Jun. 16, 1998, which is incorporated herein by reference in its entirety).
Other methods have been developed which use solid phase arrays to analyze single nucleotide polymorphisms (SNPs). For example, several primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (e.g., Kornher, J. S. et al., 1989, Nucl. Acids Res. 17:7779-7784; Sokolov, B. P., 1990, Nucl. Acids Res. 18:3671; Syvanen, A.-C. Et al., 1990, Genomics 8:684-692; Kuppuswamy, M. N. et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:11431147; Prezant, T. R. et al., 1992, Hum. Mutat. 1:159-164; Ugozzoli, L. et al., 1992, GATA 9:107-112; Nyren, P. et al., 1993, Anal. Biochem. 208:171-175; and Wallace WO89/10414).
An alternative xe2x80x9cmicro sequencingxe2x80x9d method, the Genetic Bit Analysis (GBAxe2x80x2xe2x80x3) method has been disclosed by Goelet, P. et al. (WO 92/15712). Several other micro sequencing methods have also been described, including variations of the (GBATM) 10 method of Goelet et al. (see, e.g., Mundy, U.S. Pat. No. 4,656, 127; Vary and Diamond, U.S. Pat. No. 4,851, 331; Cohen, D. et al., PCT Application No. WO 91/02087; Chee, M. et al., PCT Application No. WO 95/11995; Landegren, U. et al., 1998, Science 241:1077-1080; Nicerson, D. A. et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927; Pastinen, T. et al., 1997, Genome Res. 7:606-614; Pastinen, T. et al., 1996, Clin. Chem. 42:1391-1397; Jalanko, A. et al., 1992, Clin. Chem. 38:39-43; Shumaker, J. M. et al., PCT Application Wo 95/00669).
Other methods of nucleic acid analysis involve amplifying defined segments of nucleic acid sequence for subsequent analysis, e.g., by one or more of the micro sequencing methods discussed above. For example, the Polymerase Chain Reaction (PCR) is widely used to amplify defined segments of nucleic acid sequence in vitro. Generally, a targeted polynucleotide segment is flanked by two oligonucleotide primers. PCR consists of three steps that are repeated many times in a cyclical manner: (1) denaturing double-stranded polynucleotide sample at high temperature (about 94xc2x0 C.); (2) annealing oligonucleotide primers to the polynucleotide template at low temperature (from about 37xc2x0 C. to about 55xc2x0 C.); and (3) extending primers using a template-dependent polymerase at a moderate temperature (about 72xc2x0 C.).
PCR has been demonstrated in micro-fabricated devices consisting of a reaction chamber that has been etched into a silicon chip (see, e.g., Wilding et al., 1994, Clin. Chem. 40:1815-1818; Northrup et al., 1993, Transducers"" 93:924-926), and continuous flow PCR has been accomplished on a chip (see, e.g., Kopp et al., 1998, Science 280:1046-1048). However, these devices have the major drawback of significant solution evaporation at the high temperatures used in PCR. Further, the devices do not contain integrated micro valves or micro pumps.
A powerful concept and method known as Microfluidicsenabled Target Amplification (MeTA), which may be used as an alternative to PCR, has been previously described as set forth in U.S. patent application Ser No. 08/924,763 (Kumar, R., xe2x80x9cAMPLIFICATION METHOD FOR A POLYNUCLEOTIDE,xe2x80x9d filed Aug. 27, 1997) which is incorporated herein, by reference, in its entirety. This target amplification method is an isothermal process, using chemicals rather than high temperature DNA denaturation. MeTA method is preferably implemented using a micro-fabricated device such as the device disclosed in U.S. provisional application No. 60/110,367 (Fan, Z. H. et al., xe2x80x9cMICROFLUIDICS-BASED DEVICE FOR DNA TARGET AMPLIFICATIONxe2x80x9d filed Nov. 30, 1998) which is incorporated herein, by reference, in its entirety.
However, implementation of such methods requires special micro-fabricated devices. Currently, such micro-fabricated devices are typically fabricated from glass, silicon, or plastic plates or slides, which may be etched with horizontal or vertical cells (chambers) and/or channels, e.g., by photolithography, chemical and/or laser etching, and bonding techniques. However, such devices are fragile and expensive to produce. Alternatively, techniques have been developed for fabricating surface relief patterns in the plane of self-assembled monolayers (xe2x80x9cSAMsxe2x80x9d). These techniques typically comprise casting a material such as polydimethylsiloxane (PDMS), in the form of a prepolymer, onto a complementary relief pattern (i.e., a xe2x80x9ccastxe2x80x9d) and then curing the prepolymer (see, e.g., Wilbur et al., 1995, Adv. Mater 7:649652; Kumar et al., 1994, Langmuir 10:1498-1511; Xia Whitesides, 1995, Adv. Mater 7:471-473). The cured polymer is then used as an elastomeric stamp to form a pattern on the SAM.
However, there remains a need in the art for compositions which may be used in micro-fabricated devices for one or more bioassays such as those described above for polynucleotide analysis. In particular, such compositions should be easy to construct and relatively cheap and durable. Ideally, such compositions should also be constructed of a biocompatible material that has mechanical, optical, thermal, chemical, and electrical properties conducive to biological assays, and, in particular, to polynucleotide assays.
The present invention relates to polymer microstructures which may be used to construct or, alternatively, as part of micro-fabricated devices for biological assays. In particularly preferred embodiments the polymer microstructures of the invention are used in micro-fabricated-devices for polynucleotide analysis including, but by no means limited to, DNA micro arrays and Microfluidics devices, e.g., for PCR or MeTA polynucleotide amplification reactions.
The polymer microstructures of the invention include, but are by no means limited to, two-dimensional microstructures which may be sealed or bonded to a smooth surface such as glass. Alternatively, the two-dimensional microstructures of the present invention may also be sealed or bonded to the surfaces of other microstructures, including the surfaces of other two-dimensional polymer microstructures of this invention. Thus, the present invention also contemplates three-dimensional microstructures which may be formed by generating interlocking layers of microstructures.
The invention includes a variety of microstructures, and their use in a variety of applications for nucleotide sequence analysis, as well as for other biological applications. The microstructures of the invention include, for example, two dimensional arrays of channels and wells which may be used, e.g., to construct DNA micro arrays for polynucleotide sequence analysis. The microstructurs of the invention also include valves or pumps which comprise cavities (e.g., cells) in multilayered polymer microstructures and which may be used, e.g., to direct reagents in biological assays. The microstructures of the invention further include optical gratings, lenses, and reflective coatings, such as mirrors, which may be used, e.g., for signal detection or for sample illumination.
In preferred embodiments, the microstructures of the invention are fabricated from flexible polymer materials that include but are not limited to materials such as silicone, urethane, latex, or vinyl. The microstructures of the invention may also be fabricated from mixtures of such polymers and copolymers. In a particularly preferred embodiment, the microstructures of the invention are fabricated from polydimethylsiloxane (PDMS).
The invention is based, at least in part, on the discovery that microstructures generated by casting or molding and curing a polymer material such as PDMS onto a complementary relief pattern may be used for a variety of biological applications.
The microstructures of the present invention overcome the limitations of the prior art by providing microstructures that are durable and potentially re-usable, yet are cheap enough to be usable as disposable devices. The microstructures of the invention are also self-sealing, and able to form watertight seals on smooth surfaces by reversible bonds. Alternatively, the microstructures of the invention may be permanently bonded to another surface, e.g., by chemical treatment such as oxidative etching or use of an adhesive material. Thus, a microstructure of this invention can be readily attached, not only to surfaces such as glass or silicon, but also to the surface of another microstructure of the invention.
The microstructures of the present invention further improve upon the prior art in that they can be readily designed or modified to have particular properties which may be desirable for certain applications, e. g., in certain biological assays. For example, the surface of the microstructures may be coated or chemically modified with different chemicals or functional groups such as bovine serum albumin (xe2x80x9cBSAxe2x80x9d), glycine, polyethylene glycol (xe2x80x9cPEGxe2x80x9d), silanes, or other proteins or amino acid residues, to control surface properties, such as hydrophobicity, hydrophilicity, local surface pH, and chemical reactivity. The microstructures of the invention can also be designed to have particular bulk or regional mechanical and/or optical properties, e.g., by embedding or doping the microstructures with additional materials. For example, the microstructures of the invention can be embedded with fibers, particles and/or other polymers or copolymers to modify their mechanical properties. The microstructures of the invention may also be embedded with additives such as dyes or light absorbing or scattering particles, to modify their optical properties.
The microstructures of the invention are ideally suited for many biological applications, including assays which analyze and manipulate polynucleotide sequences. Such assays include, but are by no means limited to, assays for genetic analysis such as nucleotide sequencing assays and the detection and/or analysis of nucleotide polymorphisms such as base deletions, insertions, and SNPs. The microstructures are also well suited for in vitro biological reactions that may be performed in such assays. For example, the microstructures of the invention may be used to amplify polynucleotide sequences, e.g., by PCR or by MeTA amplification. Further, because the simple microstructures of the present invention may be combined to form more intricate, three dimensional microstructures, microstructures may also be designed and built which perform several biological reactions and/or assays simultaneously. For example, given what is taught herein, the skilled artisan may readily design and construct a microstructure which may be used, e.g., to perform sample extraction, amplify a polynucleotide sample (for example by PCR or by MeTA), and then detect SNPs in that sample on a micro array.
Biological assays and/or reactions using the microstructures of the invention require only small amounts of sample, and may be readily automated. Signal detection and analysis are also facilitated in assays using the microstructures of the invention since the optical properties of the microstructures can be adjusted to incorporate features such as optical wave guides, diffraction gratings, lenses and mirrors.
Thus, biological samples may be optically excited and signals may be directly detected within or through the microstructures, e.g., by means of an integrated device, for instance using diode lasers, total internal reflection MR), or surface plasmon resonance (SPR), CCD or other detectors, including sample imaging, for instances by lenses or lens arrays or through proximity focused detection. Alternatively, the microstructures of the invention may be readily removed, e.g., from a silicon or glass plate or slide, to expose a sample for signal detection and/or for subsequent use or analysis (e.g., in another biological assay).
The microstructures of the present invention are simple, robust, and inexpensive to produce, and may be readily customized for use in a variety of applications. Indeed, the microstructures are ideally suited for automated biological assays and robotic handling of fluids or of the microstructures, including assays for polynucleotide sequence analysis.